Isc1 regulates sphingolipid metabolism in yeast mitochondria.

1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

Abstract

The Saccharomyces cerevisiae inositol sphingolipid phospholipase C (Isc1p), a homolog of mammalian neutral sphingomyelinases, hydrolyzes complex sphingolipids to produce ceramide in vitro. Epitope-tagged Isc1p associates with the mitochondria in the post-diauxic phase of yeast growth. In this report, the mitochondrial localization of Isc1p and its role in regulating sphingolipid metabolism were investigated. First, endogenous Isc1p activity was enriched in highly purified mitochondria, and western blots using highly purified mitochondrial membrane fractions demonstrated that epitope-tagged Isc1p localized to the outer mitochondrial membrane as an integral membrane protein. Next, LC/MS was employed to determine the sphingolipid composition of highly purified mitochondria which were found to be significantly enriched in alpha-hydroxylated phytoceramides (21.7 fold) relative to the whole cell. Mitochondria, on the other hand, were significantly depleted in sphingoid bases. Compared to the parental strain, mitochondria from isc1Delta in the post-diauxic phase showed drastic reduction in the levels of alpha-hydroxylated phytoceramide (93.1% loss compared to WT mitochondria with only 2.58 fold enrichment in mitochondria compared to whole cell). Functionally, isc1Delta showed a higher rate of respiratory-deficient cells after incubation at high temperature and was more sensitive to hydrogen peroxide and ethidium bromide, indicating that isc1Delta exhibits defects related to mitochondrial function. These results suggest that Isc1p generates ceramide in mitochondria, and the generated ceramide contributes to the normal function of mitochondria. This study provides a first insight into the specific composition of ceramides in mitochondria.

WT cells were incubated to the post-diauxic phase. Sphingolipids were analyzed by LC-MS and normalized by organic phosphates as described in Materials and Methods. (A) Species which are more than 0.040 pmol/nmol organic phosphate, (B) species which are more than 0.0050 pmol/nmol organic phosphate but less than 0.040 pmol/nmol organic phophate, and (C) species which are less than 0.0050 pmol/nmol organic phosphate of the whole cell of WT. The results are mean values +/− SEM of lipid measurements from three independent cultures.

WT cells were incubated to the post-diauxic phase, and mitochondria were purified as described in Materials and Methods. Sphingolipids were analyzed by LC-MS and normalized by organic phosphates as described in Materials and Methods. (A) Species which are more than 0.040 pmol/nmol organic phosphate, (B) species which are more than 0.0050 pmol/nmol organic phosphate but less than 0.040 pmol/nmol organic phophate, and (C) species which are less than 0.0050 pmol/nmol organic phosphate of the WT mitochondria. The results are mean values +/− SEM of three lipid measurements from two independent mitochondrial purifications. Mitochondrial sphingolipids were divided by the amount of sphingolipids in the whole cell analyzed in Fig. 2. (D) Ratio of mitochondrial sphingolipids to the sphingolipids of the whole cell. Sphingolipids that were increased in mitochondria are shown. (E) Ratio of the sphingolipids in the whole cell to mitochondrial sphingolipids. Sphingolipids that were decreased in mitochondria are shown.

isc1⊿ cells were incubated to the post-diauxic phase, and collected for whole cell analyses or for mitochondrial purification as described in Materials and Methods. Sphingolipids were analyzed by LC-MS as described in Materials and Methods, and normalized by organic phosphates. (A) Species which are more than 0.040 pmol/nmol organic phosphate, (B) species which are more than 0.0050 pmol/nmol organic phosphate but less than 0.040 pmol/nmol organic phophate, (C) species which are less than 0.0050 pmol/nmol organic phosphate of isc1⊿ whole cell. (D) Species which are more than 0.040 pmol/nmol organic phosphate, (E) species which are more than 0.0050 pmol/nmol organic phosphate but less than 0.040 pmol/nmol organic phophate, and (F) species which are less than 0.0050 pmol/nmol organic phosphate of isc1⊿ mitochondria. The results are mean values +/− SEM of lipid measurements from three independent cultures for the whole cell analysis, or, three lipid measurements from two independent mitochondrial purifications for mitochondrial analysis.

The relative amounts of individual mitochondrial sphingolipids of isc1⊿ and isc1⊿+pYES/FLAG-ISC1 were compared with those of WT. (A) Ratio of the sphingolipids in WT relative to those in isc1⊿ . Sphingolipids that were decreased in isc1⊿ are shown. (B) Ratio of the sphingolipids in isc1⊿ relative to those in WT. Sphingolipids that were increased in isc1⊿ are shown. (C) Ratio of the sphingolipids in isc1⊿ and isc1⊿+pYES/FLAG-ISC1 relative to those in WT.

(A) Petite ratio of isc1⊿ . WT and isc1⊿ cells were inoculated at OD600 of 0.1 in YPD medium, incubated at 39°C for the indicated times, and plated onto YPD plates. Small colonies that grew on YPD plates were replica plated onto YPGE plates to confirm the inability to grow on non-fermentable carbon medium, and the numbers of the colonies which grew on YPD but not on YPGE plates were counted. Statistical differences were evaluated by Student's unpaired t test and are indicated by asterisks (**p<0.01, *p<0.12). (B) Hydrogen peroxide sensitivity of isc1⊿. WT and isc1⊿ were treated with 1 mM hydrogen peroxide for 45 min, plated onto YPD and the formed colonies were counted. Statistical difference was evaluated by Student's unpaired t test and is indicated by asterisks (**p<0.01). (C) Ethidium bromide sensitivity of isc1⊿. WT and isc1⊿ cells were treated with 50 μM ethidium bromide, and 5 μl of the suspension was spotted onto a YPD plate with serial dilutions at 10 fold.